Overview of Regulatory Guidelines on Impurities and Genotoxic Impurities

Medicines Agency 18 151 Genotoxic Compounds with Sufficient (Experimental) Evidence

for a Threshold-Related Mechanism 19 152 Genotoxic Compounds without Sufficient Evidence for a

Threshold-Related Mechanism 20 153 Threshold of Toxicological Concern 20

16 Questions and Answers on the “Guideline on the Limits of Genotoxic Impurities,” European Medicines Agency 23

17 Genotoxic and Carcinogenic Impurities in Drug Substances and Products: Recommended Approaches, Draft Guidance, the Food and Drug Administration 27 171 Acceptable Levels to Support Marketing Applications 33 172 Acceptable Levels during Clinical Development 35

18 Assessment and Control of DNA-Reactive (Mutagenic) Impurities in Pharmaceuticals to Limit Potential Carcinogenic Risk, ICH M7 Draft Consensus Guideline 37 181 Hazard Assessment Elements 42 182 Risk Characterization 43 183 Control 46 184 Documentation for Clinical Development Trial Application 48 185 Documentation for Common Technical Document (Marketing

Application) 49 186 Glossary 53

Impurities in drug substances are defined as “any component of the new drug substance that is not the chemical entity defined as the new drug substance” according to the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) Q3A(R2) guideline [1] In the case of drug products, impurities are defined as “any component of the new drug product that is not the drug substance or an excipient in the drug product” according to the ICH Q3B(R2) guideline [2] A subset of the impurities is genotoxic, presenting a safety concern to clinical trial subjects and patients This chapter provides an overview of the regulatory guidelines that are relevant to impurities and genotoxic impurities

The ICH [3] is a joint initiative involving both regulators and research-based industry focusing on the technical requirements for medicinal products containing new drugs The ICH brings together the regulatory authorities and pharmaceutical industry of Europe, Japan, and the United States to discuss scientific and technical aspects of drug registration Since its inception in 1990, the ICH has evolved, through its ICH Global Cooperation Group, to respond to the increasingly global face of drug development, so that the benefits of international harmonization for better global health can be realized worldwide ICH’s mission is to achieve greater harmonization to ensure that safe, effective, and high-quality medicines are developed and registered in the most resource-efficient manner The main focus of the ICH process is the preparation of harmonized guidelines that are adopted in the three ICH regions: the European Union (EU), the United States, and Japan Countries outside the ICH may also use the ICH guidelines within their own countries Generally, the ICH guidelines are accepted as the industry standard ICH guideline topics are divided

19 Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use, ICH S2(R1) 57 191 Standard Test Battery for Genotoxicity 57 192 Description of Two Options for the Standard Battery 58 193 Modifications to the Test Battery 59 194 Detection of Germ Mutations 59 195 Recommendations for In Vitro Tests 59 196 Recommended Protocols for the Mammalian Cell Assays 59 197 Recommendations for In Vivo Tests 60 198 Testing Compounds That Are Toxic for Blood or Bone Marrow 60 199 Demonstration of Target Tissue Exposure60 1910 Additional Study Design Considerations 61 1911 Guidance on Evaluation of Test Results and on Follow-Up Test

Strategies 61 1912 Evaluation of Results Obtained in In Vitro and In Vivo Tests 62 1913 Follow-Up Strategies for Positive Results 62 1914 Follow-Up Genotoxicity Testing in Relation to Tumor Findings

in a Carcinogenicity Bioassay 62 References 63

into four categories (Q, S, E, and M), and ICH topic codes are assigned according to these categories The current description of the four categories is quoted as follows:

Q (quality guidelines): harmonization achievements in the quality area include pivotal milestones such as the conduct of stability studies, defining relevant thresholds for impurities testing, and a more flexible approach to pharmaceutical quality based on good manufacturing practice (GMP) risk management

S (safety guidelines): the ICH has produced a comprehensive set of safety guidelines to uncover potential risks like carcinogenicity, genotoxicity, and reprotoxicity A recent breakthrough has been a nonclinical testing strategy for assessing the QT interval prolongation liability: the single most important cause of drug withdrawals in recent years

E (efficacy guidelines): the work carried out by the ICH under the efficacy heading is concerned with the design, conduct, safety, and reporting of clinical trials It also covers novel types of medicines derived from biotechnological processes and the use of pharmacogenetics/genomics techniques to produce better targeted medicines

M (multidisciplinary guidelines): these are the cross-cutting topics that do not fit uniquely into one of the quality, safety, and efficacy categories This category includes ICH medical terminology (MedDRA), the common technical document, and development of Electronic Standards for the Transfer of Regulatory Information

The following sections provide a summary of pertinent guidelines with the focus on aspects of impurities and genotoxic impurities In order not to change the meaning of the guidelines, the original content has been used as closely as possible References cited in the guidelines are not included in this chapter for the sake of conciseness

ICH Q3A(R2) [1] is intended to provide guidance for registration applications on the content and quantification of impurities in new drug substances produced by chemical syntheses and not previously registered in a region or member state It is not intended to apply to new drug substances used during the clinical research stage of development Although this guideline is not intended to be applied during the clinical research stage of development, in later stages of development the thresholds in this guideline can be useful in evaluating new impurities observed in the drug substance batches prepared by the proposed commercial process

Impurities in new drug substances are addressed from two perspectives:

• Chemistry aspects include classification and identification of impurities, report generation, listing of impurities in specifications, and a brief discussion on analytical procedures

• Safety aspects include specific guidance for qualifying those impurities that were not present, or were present at substantially lower levels, in batches of new drug substances used in safety and clinical studies

Impurities can be classified into the following categories:

• Organic impurities (process and drug related) • Inorganic impurities • Residual solvents

Organic impurities can arise during the manufacturing process and/or storage of the new drug substances They can be identified or unidentified, volatile or nonvolatile, and include the following:

• Starting materials • By-products • Intermediates • Degradation products • Reagents, ligands, and catalysts

Inorganic impurities can result from the manufacturing process They are normally known and identified and include the following:

• Reagents, ligands, and catalysts • Heavy metals or other residual metals • Inorganic salts • Other materials (eg, filter aids and charcoal)

Solvents are inorganic or organic liquids used as vehicles for the preparation of solutions or suspensions in the synthesis of a new drug substance Since these are generally of known toxicity, the selection of appropriate controls is easily accomplished (see ICH guideline Q3C on residual solvents [3])

Details of the actual and potential organic impurities most likely to arise during the synthesis, purification, and storage of a new drug substance should be summarized and included in the registration application The summary should be based on a sound scientific appraisal of the chemical reactions involved in the synthesis, impurities associated with raw materials that could contribute to the impurity profile of the new drug substance, and possible degradation products This discussion can be limited to those impurities that might reasonably be expected based on the knowledge of the chemical reactions and conditions involved

Table 11 shows the reporting, identification, and qualification thresholds Any impurity at a level greater than (>) the identification threshold given in Table 11 (eg, calculated using the response factor of the drug substance) in any batch manufactured by the proposed commercial process should be identified The studies conducted to characterize the structure of actual impurities present in the new drug substance at a level greater than (>) the identification threshold should be described In addition, any degradation product observed in stability studies at recommended storage conditions at a level greater than (>) the identification threshold should be identified The note in Table 11 stating that “lower thresholds can be appropriate if the impurity is unusually toxic” is applicable to genotoxic impurities

Identification of impurities present at an apparent level not more than (≤) the identification threshold is generally not considered necessary However, analytical procedures should be developed for those potential impurities that are expected to be unusually potent, producing toxic or pharmacological effects at a level not more than (≤) the identification threshold Genotoxic impurities are considered to be unusually potent impurities in this respect

Documented evidence that the analytical procedures are validated and suitable for the detection and quantification of impurities should be included in the registration application Technical factors (eg, manufacturing capability and control methodology) can be considered part of the justification for selection of alternative thresholds based on manufacturing experience with the proposed commercial process The use of two decimal places for thresholds (Table 11) does not necessarily reflect the precision of the analytical procedure used for routine quality control purposes The quantitation limit for the analytical procedure should be not more than (≤) the reporting threshold Any impurity at a level greater than (>) the reporting threshold and total impurities observed in these batches of the new drug substance should be reported with the analytical procedures indicated Below 10%, the results should be reported to two decimal places (eg, 006% and 013%); at and above 10%, the results should be reported to one decimal place (eg, 13%) Results should be rounded using conventional rules All impurities at a level greater than (>) the reporting threshold should be summed and reported as total impurities

For impurities known to be unusually potent or to produce toxic or unexpected pharmacological effects, the quantitation/detection limit of the analytical procedures should be commensurate with the level at which the impurities should be controlled Genotoxic impurities belong to this category

Acceptance criteria should be set no higher than the level that can be justified by safety data and should be consistent with the level achievable by the manufacturing process and the analytical capability Where there is no safety concern, impurity

TABLE 1.1 Reporting, Identification, and Qualification Thresholds for Impurities in New Drug Substances, ICH Q3A

acceptance criteria should be based on data generated on batches of the new drug substance manufactured by the proposed commercial process, allowing sufficient latitude to deal with normal manufacturing and analytical variations and the stability characteristics of the new drug substance

Qualification is the process of acquiring and evaluating data that establishes the biological safety of an individual impurity or a given impurity profile at the levels specified A rationale for establishing impurity acceptance criteria that includes safety considerations should be provided in the registration application The level of any impurity present in a new drug substance that has been adequately tested in safety and/ or clinical studies would be considered qualified Impurities that are also significant metabolites present in animal and/or human studies are generally considered qualified

Figure 11 describes considerations for the qualification of impurities when thresholds are exceeded In some cases, decreasing the level of impurity to not more than the threshold can be simpler than providing safety data Alternatively, adequate data could be available in the scientific literature to qualify an impurity If neither is the case, additional safety testing should be considered The studies considered appropriate to qualify an impurity will depend on a number of factors, including the patient population, daily dose, and route and duration of drug administration Such studies can be conducted on the new drug substance containing the impurities to be controlled, although studies using isolated impurities can sometimes be appropriate

ICH Q3B(R2) [2] provides guidance for registrations applications on the content and qualification of impurities in new drug products produced from chemically synthesized new drug substances This guideline is complementary to the ICH Q3A(R) guideline The ICH Q3C guideline on residual solvents should also be consulted, if appropriate

This guideline addresses only those impurities in new drug products classified as degradation products of the drug substance or reaction products of the drug substance with an excipient and/or immediate container closure system (collectively referred to as “degradation products” in this guideline) Generally, impurities present in the new drug substance need not be monitored or specified in the new drug product unless they are also degradation products Impurities arising from excipients present in the new drug product or extracted or leached from the container closure system are not covered by this guideline This guideline also does not apply to new drug products used during the clinical research stages of development

The applicant should summarize the degradation products observed during manufacture and/or stability studies of the new drug product This summary should be based on a sound scientific appraisal of potential degradation pathways in the new drug product and impurities arising from the interaction with excipients and/or the immediate container closure system In addition, the applicant should summarize any laboratory studies conducted to detect degradation products in the new drug product This summary should also include test results of batches manufactured during the development process and batches representative of the proposed commercial process

Any degradation product observed in stability studies conducted at the recommended storage condition should be identified when present at a level

greater than (>) the identification thresholds given in Table 12 When identification of a degradation product is not feasible, a summary of the laboratory studies demonstrating the unsuccessful efforts to identify it should be included in the registration application

Degradation products present at a level not more than (≤) the identification threshold would generally not need to be identified However, analytical procedures should be developed for those degradation products that are suspected to be unusually potent, producing toxic or significant pharmacological effect at levels not more than (≤) the identification threshold In unusual circumstances, technical factors (eg, manufacturing capability, a low drug substance to excipient ratio, or the use of excipients that are crude products of animal or plant origin) can be considered part of the justification for selection of alternative thresholds based on manufacturing experience with the proposed commercial process

Analytical procedures should be validated to demonstrate specificity for the specified and unspecified degradation products As appropriate, this validation should

TABLE 1.2 Thresholds for Degradation Products in New Drug Products, ICH Q3B

include samples stored under relevant stress conditions: light, heat, humidity, acid/ base hydrolysis, and oxidation The quantitation limit for the analytical procedure should be not more than (≤) the reporting threshold

The specification for a new drug product should include a list of degradation products expected to occur during manufacture of the commercial product and under recommended storage conditions Stability studies, knowledge of degradation pathways, product development studies, and laboratory studies should be used to characterize the degradation profile The selection of degradation products in the new drug product specification should be based on the degradation products found in batches manufactured by the proposed commercial process The individual degradation products with specific acceptance criteria included in the specification for the new drug product are referred to as “specified degradation products” in this guideline Specified degradation products can be identified or unidentified A rationale for the inclusion or exclusion of degradation products in the specification should be presented This rationale should include a discussion on the degradation profiles observed in the safety and clinical development batches and in stability studies, together with a consideration of the degradation profile of batches manufactured by the proposed commercial process

For degradation products known to be unusually potent or to produce toxic or unexpected pharmacological effects, the quantitation/detection limit of the analytical procedures should be commensurate with the level at which the degradation products should be controlled For a given degradation product, its acceptance criterion should be established by taking into account its acceptance criterion in the drug substance, its qualified level, its increase during stability studies, and the proposed shelf life and recommended storage conditions for the new drug product

Where there is no safety concern, degradation product acceptance criteria should be based on data generated from batches of the new drug product manufactured by the proposed commercial process, allowing sufficient latitude to deal with normal manufacturing and analytical variations and the stability characteristics of the new drug product

Qualification is the process of acquiring and evaluating data that establishes the biological safety of an individual degradation product or a given degradation profile at the levels specified The applicant should provide a rationale for establishing degradation product acceptance criteria that includes safety considerations The level of any degradation product present in the new drug product that has been adequately tested in safety and/or clinical studies would be considered qualified Therefore, it is useful to include any available information on the actual content of degradation products in the relevant batches at the time of use in safety and/or clinical studies Degradation products that are also significant metabolites present in animal and/ or human studies are generally considered qualified Degradation products could be considered qualified at levels higher than those administered in safety studies based on a comparison between the actual doses given in the safety studies and the intended dose of the new drug product

Higher or lower thresholds for qualification of degradation products can be appropriate for some individual new drug products based on scientific rationale and level of concern, including drug class effects and clinical experience For  example, qualification can be especially important when there is evidence that such degradation

products in certain new drug products or therapeutic classes have previously been associated with adverse reactions in patients In these instances, a lower qualification threshold can be appropriate Conversely, a higher qualification threshold can be appropriate for individual new drug products when the level of concern for safety is less than usual based on similar considerations (eg, patient population, drug class effects, and clinical considerations) Proposals for alternative thresholds would be considered on a case-by-case basis

Figure 12 describes considerations for the qualification of degradation products when thresholds are exceeded In some cases, reducing the level of degradation products (eg, use of a more protective container closure or modified storage conditions) to not more than (≤) the threshold can be simpler than providing safety data Alternatively, adequate data could be available in the scientific literature to qualify a degradation product If neither is the case, additional safety testing should be considered The studies considered appropriate to qualify a degradation product will depend on a number of factors, including the patient population, daily dose, and route and duration of new drug product administration Such studies can be conducted on the new drug product or substance containing the degradation products to be controlled, although studies using isolated degradation products can sometimes be appropriate

Although this guideline is not intended to be applied during the clinical research stage of development, in later stages of development the thresholds in this guideline can be useful in evaluating new degradation products observed in the new drug product batches prepared by the proposed commercial process Any new degradation product observed in the later stages of development should be identified if its level is greater than (>) the identification threshold given in Table 12 Similarly, qualification of the degradation product should be considered if its level is greater than (>) the qualification threshold given in Table 12 Safety studies should provide a comparison of the results of safety testing of the new drug product or drug substance containing a representative level of the degradation products with previously qualified material, although studies using the isolated degradation products can also be considered

The objective of Q3C (R5) [4] is to recommend acceptable amounts for residual solvents in pharmaceuticals for the safety of the patient The guideline recommends the use of less toxic solvents and describes levels considered to be toxicologically acceptable for some residual solvents

Residual solvents in pharmaceuticals are defined here as organic volatile chemicals that are used or produced in the manufacture of drug substances or excipients, or in the preparation of drug products The solvents are not completely removed by practical manufacturing techniques Appropriate selection of the solvent for the synthesis of drug substances may enhance the yield or determine characteristics such as crystal form, purity, and solubility Therefore, the solvent may sometimes be a critical parameter in the synthetic process This guideline does not address solvents deliberately used as excipients nor does it address solvates However, the content of solvents in such products should be evaluated and justified

Since there is no therapeutic benefit from residual solvents, all residual solvents should be removed to the extent possible to meet product specifications, GMPs, or other quality-based requirements Drug products should contain no higher levels of residual solvents than can be supported by safety data Some solvents that are known to cause unacceptable toxicities (class 1, Table 13) should be avoided in the production of drug substances, excipients, or drug products unless their use can be strongly justified in a risk-benefit assessment Some solvents associated with less severe toxicity (class 2, Table 13) should be limited to protect patients from potential adverse effects Ideally, less toxic solvents (class 3, Table 13) should be used where practical

TABLE 1.3 Classification of Solvents in Pharmaceutical Products, ICH Q3C

Recommended limits of class 1 and 2 solvents or classification of solvents may change as new safety data become available Supporting safety data in a marketing application for a new drug product containing a new solvent may be based on the concepts in this guideline or the concept of qualification of impurities as expressed in the guideline for drug substances (Q3A) or drug products (Q3B), or all three guidelines

Testing should be performed for residual solvents when production or purification processes are known to result in the presence of such solvents It is only necessary to test for the solvents that are used or produced in the manufacture or purification of drug substances, excipients, or drug products Although manufacturers may choose to test the drug product, a cumulative method may be used to calculate the residual solvent levels in the drug product from the levels in the ingredients used to product the product If the calculation results in a level equal to or below that recommended in this guideline, no testing of the drug product for residual solvents needs to be considered If, however, the calculated level is above the recommended level, the drug product should be tested to ascertain whether the formulation process has reduced the relevant solvent level to within the acceptable amount The drug product should also be tested if a solvent is used during its manufacture

This guideline applies to all dosage forms and routes of administration Higher levels of residual solvents may be acceptable in certain cases such as short-term (30 days or less) or topical application Justification for these levels should be made on a case-by-case basis

TABLE 1.3 Classification of Solvents in Pharmaceutical Products, ICH Q3C

The term “tolerable daily intake” is used by the International Program on Chemical Safety (IPCS) to describe the exposure limits of toxic chemicals, and “acceptable daily intake” (ADI) is used by the World Health Organization (WHO) and other national and international health authorities and institutes The new term “permitted daily exposure” (PDE) is defined in the present guideline as a pharmaceutically acceptable intake of residual solvents to avoid confusion of differing values for ADIs of the same substance

Residual solvents were evaluated for their possible risk to human health and placed into one of three classes as follows (see Table 13):

Class 1 solvents: solvents to be avoided (known human carcinogens, strongly suspected human carcinogens, and environmental hazards) The solvents in class 1 should not be employed in the manufacture of drug substances, excipients, and drug products because of their unacceptable toxicity or their deleterious environmental effects However, if their use is unavoidable to produce a drug product with a significant therapeutic advance, then their levels should be restricted as shown in Table 13, unless otherwise justified

Class 2 solvents: solvents to be limited (nongenotoxic animal carcinogens or possible causative agents of other irreversible toxicity such as neurotoxicity or teratogenicity) Class 2 solvents should be limited in pharmaceutical products because of their inherent toxicity PDEs are given to the nearest 01 mg/day, and concentrations are given to the nearest 10 ppm The stated values do not reflect the necessary analytical precision of determination Precision should be determined as part of the validation of the method

Class 3 solvents: solvents with low toxic potential (solvents with low toxic potential to humans; no health-based exposure limit is needed Class 3 solvents have PDEs of 50 mg or more per day) The solvents in class 3 may be regarded as less toxic and having lower risk to human health Class 3 includes no solvent known as a human health hazard at levels normally accepted in pharmaceuticals However, there are no long-term toxicity or carcinogenicity studies for many of the solvents in class 3 Available data indicate that they are less toxic in acute or short-term studies and negative in genotoxicity studies It is considered that amounts of these residual solvents of 50 mg/day or less would be acceptable without justification Higher amounts may also be acceptable provided they are realistic in relation to manufacturing capability and GMPs

Two options are available when setting limits for class 2 solvents: Option 1: the concentration limits in parts per million stated in Table 13 can be

used They were calculated using the following equation by assuming a product mass of 10 g administered daily:

=

× Concentration (ppm)

1000 PDE (mg/day)

Dose (g/day)

These limits are considered acceptable for all substances, excipients, or products Therefore, this option may be applied if the daily dose is not known or fixed If all excipients and drug substances in a formulation meet the limits given in option 1,

then these components may be used in any proportion No further calculation is necessary provided the daily dose does not exceed 10 g Products that are administered in doses greater than 10 g/day should be considered under option 2

Option 2: it is not considered necessary for each component of the drug product to comply with the limits given in option 1 The PDE in terms of milligrams per day as stated in Table 13 can be used with the known maximum daily dose and the aforementioned equation to determine the concentration of the residual solvent allowed in the drug product Such limits are considered acceptable provided that it has been demonstrated that the residual solvent has been reduced to the practical minimum The limits should be realistic in relation to analytical precision, manufacturing capability, and reasonable variation in the manufacturing process, and the limits should reflect contemporary manufacturing standards Option 2 may be applied by adding the amounts of a residual solvent present in each of the components of the drug product The sum of the amounts of solvent per day should be less than that given by the PDE

The method used to establish PDEs for residual solvents is presented in Section 141 Summaries of the toxicity data that were used to establish limits are published in Pharmeuropa, Vol 9, No 1, Supplement, April 1997

The Gaylor-Kodell method of risk assessment (Gaylor, D W and Kodell, R L: Linear interpolation algorithm for low dose assessment of toxic substance J Environ. Pathology, 4, 305, 1980) is appropriate for class 1 carcinogenic solvents Only in cases where reliable carcinogenicity data are available should extrapolation by the use of mathematical models be applied to setting exposure limits Exposure limits for class 1 solvents could be determined with the use of a large safety factor (ie, 10,000 to 100,000) with respect to the no-observed-effect level (NOEL) The detection and quantitation of these solvents should be by state-of-the-art analytical techniques

Acceptable exposure levels in this guideline for class 2 solvents were established by calculation of PDE values according to the procedures for setting exposure limits in pharmaceuticals (Pharmacopeial Forum, November-December 1989), and the method adopted by the IPCS for assessing the human health risk of chemicals (Environmental Health Criteria 170, WHO, Geneva, 1994) These methods are similar to those used by the US Environmental Protection Agency (EPA) (Integrated Risk Information System) and the US Food and Drug Administration (FDA) (Redbook) and others The method is outlined here to give a better understanding of the origin of PDE values

PDE is derived from NOEL, or the lowest-observed-effect level (LOEL) in the most relevant animal study, as follows:

=

×

× × × × PDE

NOEL Weight Adjustment

F1 F2 F3 F4 F5

The PDE is derived preferably from a NOEL If no NOEL is obtained, a LOEL may be used The modifying factors proposed here, for relating the data to humans, are the same kind of “uncertainty factors” used in Environmental Health Criteria

(Environmental Health Criteria 170, WHO, Geneva, 1994), and “modifying factors” or “safety factors” used in the Pharmacopeial Forum (November-December 1989) The assumption of 100% systemic exposure is used in all calculations regardless of the route of administration

The modifying factors are as follows:

F1 = a factor to account for extrapolation between species F1 = 5 for extrapolation from rats to humans F1 = 12 for extrapolation from mice to humans F1 = 2 for extrapolation from dogs to humans F1 = 25 for extrapolation from rabbits to humans F1 = 3 for extrapolation from monkeys to humans F1 = 10 for extrapolation from other animals to humans

F1 takes into account the comparative surface area: body weight ratios for the species concerned and for humans The surface area (S) is calculated as follows:

=S kM 067

where M = body mass, and the constant k has been taken to be 10 The body weights used in the equation are the ones shown in Table 14

F2 = a factor of 10 to account for variability between individuals A factor of 10 is generally given for all organic solvents, and the factor 10 is used consistently in this guideline

F3 = a variable factor to account for toxicity studies of short-term exposure F3 = 1 for studies that last at least one half-lifetime (1 year for rodents or

rabbits; 7 years for cats, dogs, and monkeys) F3 = 1 for reproductive studies in which the whole period of organogenesis

is covered F3 = 2 for a 6-month study in rodents, or a 35-year study in nonrodents F3 = 5 for a 3-month study in rodents, or a 2-year study in nonrodents F3 = 10 for studies of a shorter duration

In all cases, the higher factor has been used for study durations between the time points, for example, a factor of 2 for a 9-month rodent study

F4 = a factor that may be applied in cases of severe toxicity, for example, nongenotoxic carcinogenicity, neurotoxicity, or teratogenicity In studies of reproductive toxicity, the following factors are used: F4 = 1 for fetal toxicity associated with maternal toxicity F4 = 5 for fetal toxicity without maternal toxicity F4 = 5 for a teratogenic effect with maternal toxicity F4 = 10 for a teratogenic effect without maternal toxicity

F5 = a variable factor that may be applied if the no-effect level was not established When only an LOEL is available, a factor of up to 10 could be used depending on the severity of the toxicity

The weight adjustment assumes an arbitrary adult human body weight for either sex of 50 kg This relatively low weight provides an additional safety factor against the standard weights of 60 or 70 kg that are often used in this type of calculation It is recognized that some adult patients weigh less than 50 kg; these patients are considered to be accommodated by the built-in safety factors used to determine a PDE If the solvent was present in a formulation specifically intended for pediatric use, an adjustment for a lower body weight would be appropriate

As an example of the application of this equation, consider a toxicity study of acetonitrile in mice that is summarized in Pharmeuropa, Vol 9, No 1, Supplement, April 1997, page S24 The NOEL is calculated to be 507 mg/kg/day The PDE for acetonitrile in this study is calculated as follows:

= ×

× × × × =PDE

(507 mg/kg/day) 50 kg

12 10 5 1 1 422 mg/day

In this example,

F1 = 12 to account for the extrapolation from mice to humans F2 = 10 to account for differences between individual humans F3 = 5 because the duration of the study was only 13 weeks

F4 = 1 because no severe toxicity was encountered F5 = 1 because the no-effect level was determined

In the Glossary section of the ICH Q3C guideline, the following terms were defined that are relevant to genotoxic impurities:

Genotoxic carcinogens: carcinogens that produce cancer by affecting genes or chromosomes

LOEL: the lowest dose of substance in a study or group of studies that produces biologically significant increases in frequency or severity of any effects in the exposed humans or animals

NOEL: the highest dose of substance at which there are no biologically significant increases in frequency or severity of any effects in the exposed humans or animals

PDE: the maximum acceptable intake per day of residual solvent in pharmaceutical products

Strongly suspected human carcinogen: a substance for which there is no epidemiological evidence of carcinogenesis but there are positive genotoxicity data and clear evidence of carcinogenesis in rodents

The toxicological assessment of genotoxic impurities and the determination of acceptable limits for such impurities in active substances is a difficult issue and not addressed in sufficient detail in the existing ICH Q3X guidances The data set usually available for genotoxic impurities is quite variable and is the main factor that dictates the process used for the assessment of acceptable limits In the absence of the data usually needed for the application of one of the established risk assessment methods, that is, data from long-term carcinogenicity studies or data providing evidence for a threshold mechanism of genotoxicity, implementation of a generally applicable approach as defined by the threshold of toxicological concern (TTC) is proposed A TTC value of 15 µg/day intake of a genotoxic impurity is considered to be associated with an acceptable risk (excess cancer risk of <1 in 100,000 over a lifetime) for most pharmaceuticals From this threshold value, a permitted level in the active substance can be calculated based on the expected daily dose Higher limits may be justified under certain conditions such as short-term exposure periods

A general concept of qualification of impurities is described in the guidelines for active substances (Q3A) or medicinal products (Q3B), whereby qualification is defined as the process of acquiring and evaluating data that establish the biological safety of an individual impurity or a given impurity profile at the levels specified In the case of impurities with a genotoxic potential, determination of acceptable dose levels is generally considered a particularly critical issue, which is not specifically covered by the existing guidelines

Guideline on the Limits of Genotoxic Impurities [5] describes a general framework and practical approaches on how to deal with genotoxic impurities in new active substances It also relates to new applications for existing active substances, where assessment of the route of synthesis, process control, and impurity profile does not

provide reasonable assurance that no new or higher levels of genotoxic impurities are introduced compared to products currently authorized in the EU containing the same active substances The same also applies to variations to existing marketing authorizations pertaining to the synthesis The guideline does, however, not need to be applied retrospectively to authorized products unless there is a specific cause for concern

In the current context, the classification of a compound (impurity) as genotoxic in general means that there are positive findings in established in vitro or in vivo genotoxicity tests with the main focus on DNA-reactive substances that have a potential for direct DNA damage Isolated in vitro findings may be assessed for in vivo relevance in adequate follow-up testing In the absence of such information, in vitro genotoxicants are usually considered as presumptive in vivo mutagens and carcinogens

According to current regulatory practice, it is assumed that (in vivo) genotoxic compounds have the potential to damage DNA at any level of exposure and that such damage may lead/contribute to tumor development Thus, for genotoxic carcinogens it is prudent to assume that there is no discernible threshold and that any level of exposure carries a risk However, the existence of mechanisms leading to biologically meaningful threshold effects is increasingly acknowledged also for genotoxic events This holds true in particular for compounds interacting with non-DNA targets and also for potential mutagens, which are rapidly detoxified before coming into contact with critical targets The regulatory approach to such chemicals can be based on the identification of a critical NOEL and the use of uncertainty factors

Even for compounds that are able to react with the DNA molecules, extrapolation in a linear manner from effects in high-dose studies to very-low-level (human) exposure may not be justified due to several protective mechanisms operating effectively at low doses However, at present it is extremely difficult to experimentally prove the existence of a threshold for the genotoxicity of a given mutagen Thus, in the absence of appropriate evidence supporting the existence of a threshold for a genotoxic compound making it difficult to define a safe dose it is necessary to adopt a concept of a level of exposure that carries an acceptable risk

Guided by existing genotoxicity data or the presence of structural alerts, potential genotoxic impurities should be identified When a potential impurity contains structural alerts, additional genotoxicity testing of the impurity, typically in a bacterial reverse mutation assay, should be considered Although according to the Q3A guideline such studies can usually be conducted on the drug substance containing the impurity to be controlled, studies using isolated impurities are much more appropriate for this purpose and highly recommended

Examples of mechanisms of genotoxicity that may be demonstrated to lead to nonlinear or thresholded dose-response relationships include interaction with the spindle apparatus of cell division leading to aneuploidy, topoisomerase inhibition, inhibition of DNA synthesis, overloading of defense mechanisms, metabolic overload, and physiological perturbations (eg, induction of erythropoiesis and hyper-or hypothermia)

For classes of compounds with clear evidence for a thresholded genotoxicity, exposure levels that are without appreciable risk of genotoxicity can be established according to the procedure outlined for class 2 solvents in Q3C (see Section 14), which is derived from the NOEL, or the LOEL in the most relevant animal study using uncertainty factors

The assessment of acceptability of genotoxic impurities for which no threshold mechanisms are identified should include both pharmaceutical and toxicological evaluations

Pharmaceutical assessment: in general, pharmaceutical measurements should be guided by a policy of controlling levels to “as low as reasonably practicable” (the ALARP principle), where avoiding is not possible A justification needs to be provided that no viable alternative exists, including alternative routes of synthesis or formulations or different starting materials This might, for instance, include cases where the structure, which is responsible for the genotoxic and/or carcinogenic potential, is equivalent to that needed in chemical synthesis (eg, alkylation reactions) If a genotoxic impurity is considered to be unavoidable in a drug substance, technical efforts (eg, purification steps) should be undertaken to reduce the content of the genotoxic residues in the final product in compliance with safety needs or to a level as low as reasonably practicable Data on the chemical stability of reactive intermediates, reactants, and other components should be included in this assessment Detection and/or quantification of these residues should be done by state-of-the-art analytical techniques

Toxicological assessment: the impossibility of defining a safe exposure level (the zero risk concept) for genotoxic carcinogens without a threshold and the realization that complete elimination of genotoxic impurities from drug substances is often unachievable require the implementation of a concept of an acceptable risk level, that is, an estimate of daily human exposure at and below which there is negligible risk to human health However, these approaches require the availability of adequate data from longterm carcinogenicity studies In most cases of toxicological assessment of genotoxic impurities only limited data from in vitro studies with the impurity (eg, the Ames test and the chromosomal aberration test) are available, and thus established approaches to determine acceptable intake levels cannot be applied The calculation of “safety multiples” from in vitro data (eg, the Ames test) is considered inappropriate for justifying acceptable limits Moreover, negative carcinogenicity and genotoxicity data with the drug substance containing the impurity at low parts-per-million levels do not provide sufficient assurance for setting acceptable limits for the impurity due to the lack of sensitivity of this testing approach Even potent mutagens and carcinogens are most likely to remain undetected when tested as part of the drug substance, that is, at very low exposure levels A pragmatic approach is therefore needed that recognizes that the presence of very low levels of genotoxic impurities is not associated with an unacceptable risk

A TTC has been developed to define a common exposure level for any unstudied chemical that will not pose a risk of significant carcinogenicity or other toxic effects This TTC value was estimated to be 15 µg/person/day The TTC, originally

developed as a “threshold of regulation” at the FDA for food-contact materials, was established based on the analysis of 343 carcinogens from a carcinogenic potency database (Gold et al, Environ. Health Perspect 1984, 58, 9-319) and was repeatedly confirmed by evaluations expanding the database to more than 700 carcinogens The probability distribution of carcinogenic potencies has been used to derive an estimate of a daily exposure level (micrograms per person) of most carcinogens that would give rise to less than a 1 in 106 (1 × 10−6) upper-bound lifetime risk of cancer (“virtually safe dose”) Further analysis of subsets of high-potency carcinogens led to the suggestion of a ten-fold lower TTC (015 µg/day) for chemicals with structural alerts that raise concern for potential genotoxicity However, for the application of a TTC in the assessment of acceptable limits of genotoxic impurities in drug substances, a value of 15 µg/day, corresponding to a 10−5 lifetime risk of cancer, can be justified as a benefit exists for pharmaceuticals It should be recognized in this context that the methods on which the TTC value is based are generally considered very conservative since they involve a simple linear extrapolation from the dose giving a 50% tumor incidence (TD50) to a 1 in 106 incidence, using TD50 data from the most sensitive species and most sensitive site (several worst-case assumptions)

Some structural groups were identified to be of such high potency that intakes even below the TTC would be associated with a high probability of a significant carcinogenic risk This group of high-potency genotoxic carcinogens comprises aflatoxin-like, N-nitroso-, and azoxy-compounds that have to be excluded from the TTC approach Risk assessment of members of such groups requires compound-specific toxicity data

There may be reasons to deviate from the TTC value based on the profile of genotoxicity results Positive results from in vitro studies may only allow exemption of an impurity from limitation at TTC level if a lack of in vivo relevance of the findings is convincingly demonstrated based on a weight-of-evidence approach (see ICH S2 guidelines) This approach will usually need negative results with the impurity from some additional in vitro and/or appropriate in vivo testing

A TTC value higher than 15 µg/day may be acceptable under certain conditions, for example, short-term exposure, for the treatment of a life-threatening condition, when life expectancy is less than 5 years, or where the impurity is a known substance and human exposure will be much greater from other sources (eg, food) Genotoxic impurities that are also significant metabolites may be assessed based on the acceptability of the metabolites

The concentration limits in parts per million of a genotoxic impurity in a drug substance derived from the TTC can be calculated based on the expected daily dose to the patient using the following equation:

Concentration (ppm)

TTC ( g/day)

Dose (g/day) =

µ

The TTC concept should not be applied to carcinogens where adequate toxicity data (long-term studies) are available and allow for a compound-specific risk assessment It has to be emphasized that the TTC is a pragmatic risk management tool using a probabilistic methodology, that is, there is a high probability that a 10−5 lifetime cancer risk will not be exceeded if the daily intake of a genotoxic impurity with

unknown carcinogenic potential/potency is below the TTC value The TTC concept should not be interpreted as providing absolute certainty of no risk

The decision tree for assessing the acceptability of genotoxic impurities is shown in Figure 13

The aim of this questions and answers document [6] is to provide clarification and harmonization of the Guideline on the Limits of Genotoxic Impurities published in 2006 Question 1: The guideline does not need to be applied retrospectively to authorized

products unless there is a specific cause for concern What might constitute a cause for concern in terms of application to currently marketed products?